Meiotic pairing is essential for proper segregation of homologs and production of chromosomally normal gametes. Pairing abnormalities cause homolog nondisjunction, resulting in aneuploidy, a major cause of spontaneous abortion and developmental abnormalities in humans. The proposed experiments utilize Drosophila male meiosis as a model system to explore how homologs align in early meiosis, how they develop cohesive bonds stable enough to resist spindle forces and how those bonds are rapidly broken when homologs segregate at anaphase. The experiments build on the prior identification of a 240bp repeated sequence located in the intergenic spacer of the rRNA genes on the X and Y chromosomes that functions as the primary X-Y meiotic pairing site. In the previous funding period we demonstrated that an RNA polymerase I promoter located in each repeat is essential for meiotic pairing. We also generated a large collection of novel meiotic mutations that define nine genes (pairing failure (pf) 1-9) needed for proper meiotic segregation of all chromosomes. We discovered that one of the genes identified in this screen (pf-1) encodes a novel member of the stromalin family of cohesin proteins (dSA-2) that we postulate to function specifically in homolog pairing. Aim 1 of this proposal is a further exploration of the link between transcription and pairing, examining both the role of rDNA transcripts in X-Y pairing, and the role of transcription generally in pairing. In aim 2 we will further characterize the phenotypes of pf mutations, focusing particularly on the kinetics of pairing failure in. male meiosis, and on possible roles in other pairing pathways in female meiosis and in somatic tissues. Aim 3 will be a comprehensive molecular analysis of the dSA-2 protein, focused especially on testing the hypothesis that it is a meiotic cohesin with a specialized role in homolog pairing. In aim 4, we propose to identify and analyze additional genes involved in male meiotic homolog pairing. Taken together, these experiments are expected to advance our understanding of chromosome pairing in Drosophila and to provide models for pairing mechanisms that can be tested in mammals.